Anita L. Zimmerman - US grants

Cyclic nucleotide-gated (CNG) ion channels are key players in the early stages of vision. They are participants in an enzyme cascade in rods and cones that converts the energy of absorbed light into an electrochemical signal to be relayed to the brain. The channels are opened by the direct binding of cyclic guanosine monophosphate (cGMP), whose free concentration is higher in the dark than in the light. Closure of the channels in the light causes a change in membrane potential that reduces the release of neurotransmitter onto second order neurons. The apparent affinity of the CNG channels for cGMP has been found to be modulated by cytosolic and membrane factors, including Ca2+/calmodulin, phosphatases and diacylglycerol. There is also evidence that the concentration and/or effectiveness of some modulatory factors depends on the amount of light absorbed. Functional modulation of the CNG channels has potential significance in two ways: 1) it may be involved in some aspect of normal visual transduction, such as in light or dark adaptation; and 2) its pharmacologic or genetic manipulation may be useful in treating some forms of retinal degeneration in which either the channel or the cGMP concentration is abnormal. CNG channels are composed of at least two kinds of subunits, alpha and beta, both of which contain cyclic nucleotide binding sites. Ca2+/calmodulin has been found to act only on the beta subunit, but it is not known which subunit(s) are controlled by the other modulators. The long-range goals of this project are to understand how rod and cone CNG channels work and how they participate and are controlled in visual transduction. The specific aims are to use electrophysiology and molecular biology techniques to study functional modulation of rod CNG channels by Ca2+/calmodulin and phosphorylation. Initially, modulation by phosphorylation will be studied in native channels from amphibian rods to determine the roles of specific phosphatases and kinases. The Xenopus oocyte expression system and patch clamp studies will allow functional analysis of wild-type cloned bovine channels as well as mutants designed to dissect the molecular mechanisms of the two forms of modulation.

The Graduate Program in Molecular Pharmacology & Physiology is a small and very diverse program with a strong set of courses and activities, as well as extensive mentoring mechanisms. Leadership of the program is by a group of three accomplished MPIs with complementary strengths and expertise that guarantee effective oversight of the program. The request is for 4 T32 slots per year; the total number of students in the program is 20. This training program in Interdisciplinary Training in Pharmacological Sciences will replace our current NIGMS pharmacology T32, ending in June of 2021. The training in our program focuses on fundamental principles of pharmacology, rigor and reproducibility, and cutting-edge quantitative methods. Our trainees participate in many activities that promote interaction, collaboration and professional development. Here we propose a number of new initiatives in the curriculum, professional development, program oversight and recruitment. A recent innovative initiative we are developing in the program is the establishment of summer internships at Pfizer pharmaceutical company labs; these internships will give our trainees the unique opportunity to experience first-hand the scientific environment in a pharmaceutical company and make informed decisions for their future careers. Our current admissions and recruitment practices have yielded a high percentage of students from disadvantaged and underrepresented groups (in the last 5 years, 47% underrepresented minorities plus 7% disabled), and there has been 100% retention of these students in the program during the last 15 years (retention for the program in general is 95%). The career outcomes of the students are excellent, with 52% of students staying in academia (25% faculty, 27% currently postdocs) often at top institutions, about 25% as scientists in pharmaceutical or biotechnology companies, and the remainder in a variety of science and medicine- related careers. Thus, our program has been training a highly diverse group of students who have become successful in different areas of science; the new initiatives proposed in our T32 application will further enhance our unique program and improve the quality of the training.